Protocol for the quantification of RNA. Provides RNA sample concentration

Qubit RNA HS Assay Kit (Qubit RNA HS Assay Kit, ThermoFisher, Catalog # Q32852)

• Qubit RNA HS Assay Kit Manufacturer’s Instructions
• “The Qubit RNA HS Assay Kit, when used with the Qubit Fluorometer, provides an accurate and selective method for the quantitation of low-abundance RNA samples. The assay is highly selective for RNA and will not quantitate DNA, protein, or free nucleotides. Common contaminants, such as salts, free nucleotides, solvents, detergents, or protein, are well-tolerated in the assay. The assay kit is designed to be accurate for RNA sample concentrations between 250 pg/µL and 100 ng/µL”

Protocol for Quantification of RNA

• Use only thin-wall, clear, 0.5-mL PCR tubes. Acceptable tubes include Qubit assay tubes (Catalog # Q32856) or Axygen PCR-05-C tubes (Catalog # 10011-830).
• If approximate sample concentration is unknown prior to measurement, run a dilution series with 1-2 samples to bring concentration within range of the assay (e.g., 1:1, 1:10, and 1:100)
• record any dilutions done prior to measurement in order to back calculate original concentration

Procedure

• Label tubes for each of your samples and 2 standards (Label lids only!)
  • High Sensitivity (standard 1 = 0 ng/μL RNA in TE buffer, standard 2 = 10 ng/μL RNA in TE buffer)
• Prepare the Qubit working solution by diluting the Qubit RNA HS Reagent 1:200 in Qubit RNA HS Buffer (Volume = #samples + #standards + 1 extra)
  • The final volume in each tube must be 200 μL
  • Each standard tube requires 190 μL of Qubit working solution, and each sample tube requires anywhere from 180–199 μL
  • Prepare sufficient Qubit working solution to accommodate all standards and samples
• Add 190μL of working solution to each of the tubes used for standards
• Add 10μL of each standard to the appropriate tube, then mix by vortexing for 2–3 seconds (Take care NOT to create bubbles)
• Add Xµl of working solution to individual assay tubes so that the final volume in each tube after adding sample is 200 μL (no less than 180µl)
• Add 200µl - Xµl of sample to each tube, then mix by vortexing for 2–3 seconds (Take care NOT to create bubbles)
• Incubate tubes at room temperature for 2min (cover with foil)
• Read standards on Qubit
• Set volume of sample and readout in ng/µl and read each tube